Stability toward protease

After immobihzation, the resistance toward protease increases. The reason may be an increase of steric access hindrance to the immobilized protease due to the three-dimensional obstacles of the carrier. If we take aminoacylase for example, under the action of trypsin, the activity of the solution enzyme was only 23%, the activity of the enzyme immobilized by DEAE-cellulose was 33% and the activity of the enzyme immobilized by DEAE-dextran was 87%. Under the action of protease, Pronase P, the activity of the solution enzyme, the enzyme immobilized by DEAE-ceUulose and the enzyme immobilized by DEAE-dextran was 48%, 53%, 88%, respectively. Immobilized protease can generally avoid digestion damaging itself. 

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Silva-Cnnha, A. et al., W/O/W multiple emulsions of insulin containing a protease inhibitor and an absorption enhancer preparation, characterization and determination of stability towards proteases in vitro, Int. J. Pharm., 158, 79,1997. 

Little was done in the area of cross-linked enzyme crystals over the next 10 years. In 1977, the kinetic properties of CLCs of the protease subtilisin were reported by Tuchsen and Ottesen [3], They reported that cross-linked enzyme crystals of subtilisin were highly effective catalysts with increased thermal stability and increased stability toward acid compared to the soluble enzyme. They further reported that the CLCs of subtilisin showed essentially no autodigestion at 30°C. Like Quiocho and Richards before them, Tuchsen and Ottesen found... 

The prehminary data suggests that aPNAs are stable towards degradation by the proteases present in human serum. Further studies are required to determine the stability of aPNAs in vivo. 

However, there are two problems with these unconjugated lactones lack of selectivity and limited stability of the inhibitor in biological buffers. Coumarin carboxylates have been developed to improve selectivity toward a given serine protease (Section 11.4.1). On the other hand, the amide bond is chemically and enzymatically much more stable than the ester one. This raised the question of whether a starting functionalized lactam behaved like the previous lactones and generated in situ a quinonimine methide, the aza-analogue of the quinone methide (Section 11.5). 

Unlike many other enzymes, the subtilisins are fairly stable towards e.g. organic solvents, anionic surfactants, high temperatures and high pH. This makes the subtilisins very suitable as detergent proteases. But despite this fact, stabilization of these protease enzymes in liquid detergents remains a major issue. 

Replacement of some amino acids in a peptide by an a-trifluoromethyl amino acid influences the stability of the peptide toward different proteases (e.g., a-chymotrip-sins). Thus, for steric reasons, introducing a trifluoromethyl group in the P] position... 

Purification of bioproducts from fermentation, although necessary, comes with a major drawback. Biological compounds evolved in an environment of complex components are adapted to be most stable in these conditions. Highly pure forms of bioproducts have a tendency towards lower chemical and physical stability. For instance, proteases are prone to autolysis in the absence of impurities that inhibit degradation. This is one of the major... 

Figure 7. Complete proteolytic stability of all types of P-and y-peptides towards a variety of peptidases. The P-peptides ranged in size from dimer to ISmer. The enzymes include all common types of peptidases (endo/exo, metallo, serine, threonine, and aspartyl proteases). After 40 hours there was no observable cleavage of any of the homologated peptides and no inhibition of the enzymes [41].

Generally, sodium and potassium react only to a limited extent with proteins, whereas calcium and magnesium are somewhat more reactive. Transition metals, e.g., ions of Cu, Fe, Hg, and Ag, react readily with proteins, many forming stable complexes with thiol groups. Calcium cations and ferrous, cupric, and magnesium cations may be integral parts of certain protein molecules or molecular associations. Their removal by dialysis or sequestration appreciably lowers the stability of the protein structure toward heat and proteases. 

The isothiazol-3(2//)-one 1,1-dioxides 254-257 with stabilizing aryl substituents in the 2-, 4- and/or 5-position are potential inhibitors toward human leukocyte elastase (HLE) (03ZN(B)111, 05JEIMC341). HLE is a serine protease implicated in several inflammatory diseases and represents a major target for the development of low-molecular weight inhibitors. 

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